METHODOLOGY Open Access Establishment of an immortalized ... · Establishment of an immortalized...

METHODOLOGY Open Access

Establishment of an immortalized humanendometrial stromal cell line with functionalresponses to ovarian stimuliMunehiro Yuhki1*, Takashi Kajitani2, Takakazu Mizuno1, Yuko Aoki1 and Tetsuo Maruyama2

Abstract

Studies on the mechanisms of decidualization and endometriosis are often hampered by lack of primaryendometrial cells. To facilitate in vitro studies, we established a human endometrial stromal cell line, KC02-44D,immortalized with human telomerase reverse transcriptase. Upon exposure to ovarian stimuli, KC02-44D cellsshowed similar cytoskeletal marker or gene expression and biochemical phenotype to primary endometrial stromalcells. KC02-44D would be useful for studies of human endometrial function and its associated pathologies.

BackgroundDecidualization is the process of differentiation of theendometrium, the mucosa lining the uterine lumen. Inendometrial decidualization, estrogen receptor (ER) andprogesterone receptor (PR) expressions strictly regulatethe tissue responsiveness to cognate ligands. Estrogencan autoregulate ER expression, and PR is a classicalestrogen target [1-3]. In humans, decidualization occursindependently of a blastocyst signal during the secondhalf of the menstrual cycle and is controlled as a proges-terone- and 3’,5’-cyclic adenosine monophosphate(cAMP)-dependent event. Its initiation requires elevatedintracellular cAMP levels and sustained activation of theprotein kinase A (PKA) pathway; progestin and cAMPsynergistically induce decidualization in primary cul-tured human endometrial stromal cells (ESCs) [4].Decidual cells exhibit morphological changes and abun-dant secretion of decidualization enhancers such as pro-lactin and insulin-like growth factor-binding protein-1(IGFBP-1) [4].Endometriosis is the growth of endometrial epithelial

and stromal cells at ectopic sites outside the uterine cav-ity. Interleukin (IL)-1b, a proinflammatory cytokine, isconsidered to play an important role in the pathogenesisof endometriosis [5,6]. However, the relationship

between IL-1b and abnormal growth of endometrialcells is not clearly understood.For investigations on the mechanisms of decidualiza-

tion and endometriosis, cultured human ESCs are desir-able tools; however, the applicability of these cells islargely restricted by the long cell-culture time and diffi-culty in maintaining the primary ESC phenotype. Severalattempts have been made to immortalize human ESCsby oncogenic transformation [7] or prolongation of celldivision by introducing human telomerase reverse tran-scriptase (hTERT) [8-11]. However, the synergistic effectof progestin and cAMP on the decidualization capacityand the induction of the PR by estradiol (E2) have notbeen fully reproduced in these cell lines. In this study,we established an hTERT-immortalized ESC line, KC02-44D, to facilitate in vitro studies. KC02-44D cellsresponded in a similar manner to primary human ESCs,showing the synergistic induction of decidualization byprogestin and cAMP, and PR upregulation by E2.

MethodsHuman tissue samplesEndometrial tissue was obtained from the uterus of a45-year-old woman who had undergone total hysterect-omy because of leiomyoma, after her written informedconsent and Keio University Hospital approval. Primaryhuman ESCs were purified as described previously [12].In brief, the tissue specimens were washed in Dulbecco’smodified Eagle’s medium (DMEM; Sigma-Aldrich, St.Louis, MO) and minced with scissors into pieces less

* Correspondence: [email protected] Research Laboratories, Chugai Pharmaceutical Co., Ltd., 200Kajiwara, Kamakura, Kanagawa 247-8530, JapanFull list of author information is available at the end of the article

Yuhki et al. Reproductive Biology and Endocrinology 2011, 9:104http://www.rbej.com/content/9/1/104

© 2011 Yuhki et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction inany medium, provided the original work is properly cited.

mailto:[email protected]

http://creativecommons.org/licenses/by/2.0

than 1 mm3 in size. The tissues were then gently agi-tated in tubes for 2 h at 37°C in DMEM with 0.2% (wt/vol) collagenase (Wako Pure Chemical Industries,Osaka, Japan), 0.05% DNase I (Life Technologies, Inc.,Gaithersburg, MD), 10% fetal bovine serum (FBS), and1% antibiotic-antimycotic mixture (Life Technologies,Grand Island, NY). After enzymatic digestion, cellclumps were dispersed by pipetting. Most of thedigested human ESCs, presenting as single cells or smallaggregates, were filtered sequentially through a 70 μmcell-strainer nylon filter (Falcon 2350; BD Biosciences,Franklin Lakes, NJ) to remove gland cells, layered ontoFicoll-Paque (GE Healthcare Life Sciences, Uppsala,Sweden), and centrifuged at 500 × g for 15 min at 4°Cto remove erythrocytes. The isolated human ESCs werecollected from the Ficoll interface and resuspended inDMEM with 10% FBS and 1% antibiotic-antimycoticmixture.

Recombinant lentivirus preparationsRecombinant lentiviruses were prepared using the Vira-Power Lentiviral Expression System (Life Technologies),according to the manufacturer’s instructions. Full-lengthcDNA of human telomerase reverse transcriptase isoform 1(hTERT; GenBank accession number NM_003219) wasamplified by PCR from the total RNA of a colon cancercell line. The first-strand DNA was synthesized usinghTERT 3’-noncoding primer 5’-TGACAGGGCTGCTGGTGTCTG and ReverTra Ace reverse transcriptase sys-tem (Toyobo, Tokyo, Japan). The cDNA of the N-termi-nus half of hTERT (1-2304) was amplified using forwardprimer 5’-CACCATGCCGCGCGCTCCCCGCTGCCGAand reverse primer 5’-GCCTTCTGGACCACGGCA-TACCGA, and that of the C-terminus half (1977-3399)was amplified using forward primer 5’-CACCGG-CACTGTTCAGCGTGCTCAACTACGAG and reverseprimer 5’-TCAGTCCAGGATGGTCTTGAAGTC. EachcDNA was ligated to the pLenti6/V5-D TOPO vector.Kozak sequences were introduced according to theinstruction manual. The fragment between SpeI (in thevector) and EcoRV sites from the N-terminus half wasthen recloned in the same sites of the C-terminus half toobtain the full-length hTERT plasmid (pLenti6-hTERT).The DNA sequence of pLenti6-hTERT was completelyconfirmed by DNA sequence analysis. pLenti6-hTERTwas then transfected to near-confluent 293FT cells (LifeTechnologies) with helper plasmids (pLP1, pLP2, andpLP/VSVG) to produce a lentivirus encoding hTERT(LtV-hTERT). After 2 days of incubation at 37°C, the con-ditioned mediums of the lentivirus-producing 293FT cellswere harvested and centrifuged at 3000 rpm for 15 min at4°C. The supernatants were separated into aliquots andstored at -80°C until use.

Immortalization of human ESCsFor immortalization, human ESCs were infected withLtV-hTERT in the presence of polybrene (6 mg/ml,Sigma). The cells were grown in phenol red-free DMEMsupplemented with 10% dextran-coated charcoal-treatedFBS (DCC-FBS; Life Technologies) in the presence of 10nM 17-b-estradiol (E2; E8875, Sigma) for 7-8 days untiluninfected cells died in the presence of 2 mg/ml blastici-dine (Life Technologies). Then, the cells were trypsinizedand limiting-diluted in 96-well plates. Immortalizedclones were obtained in the presence of E2 under selec-tion with blasticidine. The obtained clones were main-tained in DMEM supplemented with 10% FBS, 100 U/mlpenicillin, 100 mg/ml streptomycin, and 2 mg/ml blastici-dine at 37°C under 5% CO2 in a humidified incubator.The clones were tested for marker expression andresponse to ovarian steroids and cAMP, as describedbelow. Finally, KC02-44D was selected as the mostresponsive clone to the ovarian stimuli. We did notobtain any clones responsive to these stimuli when pri-mary human ESCs were cultured in the absence of E2.

In vitro decidualization and cytokine assayKC02-44D cells were precultured in phenol red-freeDMEM supplemented with 10% DCC-FBS, 100 U/mlpenicillin, and 100 mg/ml streptomycin at 37°C over 4days. They were then stimulated with or without 10 nME2, 1 μM 6a-methyl-17a-hydroxy-progesterone acetate(MPA; M-1629, Sigma), and/or 0.125 mM dibutyrylcAMP (D0627, Sigma) in combination with the indi-cated concentrations of cytokines for different time peri-ods according to the experimental procedures. Cellimages were acquired using a phase-contrast microscope(Nikon). In the IL-1b experiment, KC02-44D cells weretreated with 1.0 ng/ml IL-1b (201-LB-005, R&D Sys-tems, Minneapolis, MN) for 3-7 days, as described inthe figure legends.

ImmunocytochemistryKC02-44D cells were cultured in 96-well tissue cultureplates and characterized by immunofluorescence staining.The cells were fixed with 4% paraformaldehyde (PFA;Wako Pure Chemical Industries) solution for 30 min atroom temperature. After washing and permeabilizationwith phosphate-buffered saline (PBS) containing 0.1%Triton X-100, the cells were blocked with PBS-5% FBS at4°C. The fixed cells were incubated in PBS-10% FBS con-taining Cy3-labeled mouse anti-vimentin (C9080, 1:200;Sigma), mouse anti-CD10 (clone SS2/36, 1:25; Dako,Glostrup, Denmark), mouse anti-CD13 (clone WM-47,1:50; Dako), or mouse anti-cytokeratin (clone MNF116,1:50; Dako) antibody. After washing in PBS, the cellswere incubated with Alexa Fluor 488-labeled anti-mouse


of 9

http://www.ncbi.nih.gov/entrez/query.fcgi?db=Nucleotide&cmd=search&term=NM_003219

secondary antibody (1:500; Molecular Probes, Eugene,OR), washed again, and examined for each markerexpression under a fluorescence microscope (BZ-8000;Keyence, Osaka, Japan).

Western blot analysisTotal proteins from KC02-44D cells were extracted withCell Lysis Buffer (Cell Signaling Technology, Beverly,MA) containing 1 mM phenylmethanesulfonyl fluoride(Sigma) on ice. The lysate was scraped and sonicated for5 s by using an ultrasonic disruptor at output 1 (TomyDigital Biology, Tokyo, Japan). The sonicate was centri-fuged at 15,000 rpm for 10 min at 4°C, and the proteincontents of the supernatant were measured using theBradford protein assay (Bio-Rad, Hercules, CA). Afterthe addition of lithium dodecyl sulfate sample buffer(Life Technologies, Grand Island, NY) containing 2-mer-captoethanol (Sigma), the samples were reduced at 70°Cfor 10 min. Equal amounts of total proteins were thenelectrophoresed on 4-12% NuPAGE Bis-Tris gels (LifeTechnologies, Grand Island, NY) and transferred ontopolyvinylidene difluoride membranes (Immobilon-P;Millipore, Bedford, MA), followed by blocking with 5%nonfat dry milk in Tris-buffered saline containing 0.1%Tween-20 (TBS-T). The membranes were then immu-noblotted with the primary antibody against thePR (clone PgR636, 1:800, mouse monoclonal; Dako),IGFBP-1 (06-106, 1:1000, rabbit polyclonal; Upstate,Millipore), or b-actin (1:3000, goat polyclonal; SantaCruz Biotechnology, Santa Cruz, CA) in TBS-T for 1 hat room temperature. After washing thrice with TBS-T,the membranes were further incubated with horseradishperoxidase-conjugated anti-mouse, anti-rabbit (1:10,000;GE Healthcare, Buckinghamshire, UK) or anti-goat(1:5000; Jackson ImmunoResearch Laboratories, WestGrove, PA) secondary antibody in TBS-T for 1 h atroom temperature. After washing thrice with TBS-Tagain, proteins were detected with ECL Plus reagent(GE Healthcare) and visualized with LumiImager (RocheDiagnostics, Penzberg, Germany).

Enzyme-linked immunosorbent assay (ELISA)KC02-44D cells were plated at a density of 2 × 105 cellsper well in 6-well plates. Two days later, the mediumwas changed and the cells were stimulated with E2,MPA, and dibutyryl cAMP alone or in combination atthe indicated concentrations. The supernatants werecollected 6 or 7 days later, and the amounts of IGFBP-1,PGE2, and IL-8 proteins were measured using ELISAkits for IGFBP-1 (DY871, R&D Systems), prostaglandinE2 (PGE2; KGE004, R&D Systems), and IL-8 (GEHealthcare), respectively. Each treatment was performedin triplicate wells.

Reverse transcription polymerase chain reaction (RT-PCR)Total RNA was extracted from cultured KC02-44D cellsby using an RNeasy mini kit (Qiagen, Valencia, CA).RT-PCR was carried out with 200 ng total cellular RNAby using a One-Step RT-PCR kit (Qiagen). The primersused for amplification were as follows: human estrogenreceptor-alpha (ERa), forward primer 5’-CAGGGGT-GAAGTGGGGTCTGCTG-3’ and reverse primer 5’-ATGCGGAACCGAGATGATGTAGC-3’; cyclooxygen-ase-2 (COX-2), forward primer 5’-TGTCTTGACATCCAGATCAC-3’ and reverse primer 5’-ACATCATGTTTGAGCCCTGG-3’; b-actin, forward primer 5’-CCCAGGCACCAGGGCGTGATC-3’ and reverse primer5’-TCAAACATGATCTGGGTCAT-3’. Preliminaryexperiments determined the optimum PCR cycle num-ber within the linear range of amplification for eachgene being measured. After PCR amplification, the sam-ples (15 μl aliquots) were electrophoresed in 2% agarosegels, followed by photographic recording of ethidiumbromide-stained gels with FAS-III MINI (Toyobo). Alldata from the RT-PCR analysis represent the results ofthree independent experiments.

Cell-proliferation assayCell proliferation was determined using a Click-iT EdUAlexa Fluor Imaging kit (Life Technologies, Grand Island,NY), a modified 5-bromo-2’-deoxyuridine (BrdU) systemto detect DNA replication. In brief, KC02-44D cells (1.0-1.5 × 103) were seeded onto 96-well culture plates withphenol red-free DMEM containing 10% DCC-FBS and sti-mulated with cytokines 48 h later. EdU (5-ethynyl-2’-deox-yuridine) was added to a final concentration of 10 μM inthe cell-culture medium and the cells were incubated for24 h at 37°C. After fixation with 4% PFA and permeabili-zation with 0.1% Triton X-100 in PBS, the cells were trea-ted with the reaction cocktail containing Alexa Fluor 488azide. The EdU intensity was measured using an ArrayS-can VTi HCS reader (Thermo Scientific Cellomics, Pitts-burg, PA). The colocalization bioapplication protocol wasused to acquire images with the appropriate filter sets intwo separate fluorescence channels: channel 1, Hoechst33342 (nuclear reference and object identification); chan-nel 2, EdU labeled with Alexa Fluor 488 azide (identifica-tion of DNA-replicating S-phase cells in the cell cycle).The cells were imaged with a 20 × objective and 0.63numerical aperture with exposure times of 0.2 s (Hoechst33342) and 0.1 s (Alexa Fluor 488 azide). The images werecollected as nine fields per well, and the values wereexpressed as the mean average fluorescence intensity.

Statistical analysisAll quantitative experiments were conducted at leastthree times each in triplicate wells. The results were


of 9

then analyzed using ANOVA and Tukey’s test. The datashown in figures was the Mean ± SEM for triplicatewells from one representative experiment.

ResultsCharacterization of KC02-44D cellsImmunocytochemical analysisTo assess specific antigen marker expression, KC02-44Dcells were fixed, permeabilized, and immunostained withantibodies to endometrial stromal marker proteins. Thecells were positively stained with the stromal cell mar-kers vimentin, CD10, or CD13, but not with the epithe-lial cell marker cytokeratin (Figure 1).Responses to ovarian steroids and cAMPThe decidualization capacity of KC02-44D cells againstovarian steroids and cAMP was next examined. Decid-ualized stromal cells are known to change into a roundmorphology upon treatment with cAMP and a spindle-shaped morphology upon treatment with MPA [9,10].When KC02-44D cells precultured in the E2-free med-ium were treated with E2, MPA, and dibutyryl cAMPfor 6 days, they transformed into a decidualized mor-phology (Figure 2a).KC02-44D cells were also tested for their response to

ovarian steroids and dibutyryl cAMP to induce IGFBP-1, a decidualization marker [4]. MPA or dibutyryl cAMPsignificantly but very weakly induced IGFBP-1 expres-sion in the presence of E2. However, the protein expres-sion was greatly induced by the combination of MPA,dibutyryl cAMP, and E2 (Figure 2b), consistent with thefindings of a previous study on primary human ESCs[13]. Prolactin, another decidualization marker, showed

similar responses to IGFBP-1 (See Additional file 1 Fig-ure S1). KC02-44D cells maintain their response toestradiol, progestin, and cAMP even at high passagenumbers (See Additional file 2 Figure S2).We then assessed PR expression under stimulation by

ovarian steroids. Exposure of KC02-44D cells to E2strongly upregulated the expression of both isoforms (Aand B) of PR, a target gene of ERa in the endometrium[14-18]. The E2-induced PR expression was significantlydownregulated after stimulation with MPA (Figure 2c),consistent with reports on the endometrial decidualiza-tion phase [7,13,19]. Therefore, KC02-44D cells wereconsidered to possess ESC characteristics, displayingsimilar biochemical properties as well as cytoskeletalmarker and gene expression patterns to primary ESCs,without any structural abnormalities.

Responses of KC02-44D cells to IL-1bRegulation of inflammatory responses by IL-1bPro-inflammatory cytokine concentrations in peritonealfluid increase in women with endometriosis [20-22], andIL-1b is suggested to play an important role in thepathogenesis of endometriosis [5,6]. Therefore, we inves-tigated the effect of IL-1b on the inflammatoryresponses in KC02-44D cells. When the cells were trea-ted with IL-1b, COX-2 gene expression was dramaticallyenhanced (Figure 3a), and PGE2 and IL-8 secretion wasinduced (Figure 3b and 3c). COX-2 and PGE2 inductionwas canceled by the addition of MPA. In contrast, IL-8induction by IL-1b could not be suppressed by MPA,suggesting PR-independent regulation. The vehicle con-trol yielded the same result as E2 (Figure 3b and 3c).

Figure 1 Expression of marker proteins in KC02-44D cells. PFA-fixed KC02-44D cells at passage number 10 were stained with antibodiesagainst vimentin (red) as well as CD10, CD13, and cytokeratin (green, with Hoechst blue for nuclear staining). No signal was detected in thenegative controls (no antibody; lower panels). Scale bars = 100 μm.


of 9

Downregulation of decidualization capacity by IL-1bReportedly, stromal cells from endometriotic lesions andthe endometrium in women with endometriosis showreduced decidualization capacity [23], and IL-1b inhibitscAMP-mediated decidualization in primary human ESCs[24-26]. Therefore, we tested whether IL-1b can reducethe decidualization capacity of KC02-44D cells. IL-1bdrastically inhibited MPA- and dibutyryl cAMP-inducedIGFBP-1 expression (Figure 4a); moreover, the E2-induced PR expression was significantly downregulated

after incubation with IL-1b (Figure 4b). The vehicle con-trol yielded the same result as E2 for IGFBP-1expression(Figure 4a and 4b).Counteraction of ovarian steroid- and cAMP-inducedgrowth arrest by IL-1bWe used the EdU assay to assess whether IL-1b cancounteract MPA- and dibutyryl cAMP-induced growtharrest of KC02-44D cells. In the absence of the ovariansteroids and dibutyryl cAMP, IL-1b slightly enhancedEdU incorporation to DNA, indicating the induction of

Figure 2 Induction of decidualization in KC02-44D cells. KC02-44D cells were treated with the indicated combinations of 10-8 M E2, 10-6 MMPA, and 0.125 mM dibutyryl cAMP for 6 days to assess their decidualization capacity. (a) Phase-contrast microphotographs of living KC02-44Dcells at passage number 17 (scale bars = 100 μm). (b) Expression of decidualization marker IGFBP-1 in KC02-44D cells at passage number 15(ELISA). The data represent the mean ± SEM of triplicate measurements. *P < 0.05 and ***P < 0.001 by Tukey’s test. (c) Expression of the ER(RT-PCR analysis) and PR (Western blot analysis) in KC02-44D cells at passage numbers 15 and 16. b-Actin was used for normalization.


of 9

cell proliferation (Figure 5). Along with the decidualiza-tion mediated by MPA and/or dibutyryl cAMP, the cellgrowth was significantly inhibited, observed as a reduc-tion in the number of EdU-labeled S-phase cells (Figure5, CTRL). In contrast, IL-1b treatment counteracted cellgrowth arrest even in the presence of MPA and/or dibu-tyryl cAMP (Figure 5, IL-1b), indicating the inhibition ofthe decidualization capacity of KC02-44D cells.

DiscussionWe established the KC02-44D cell line and found simi-lar characteristics to primary human ESCs, showing thesynergistic induction of decidualization by progestin andcAMP. Moreover, our cell line showed upregulation ofPR expression upon E2 stimulation, which is critical forthe progestin-dependent decidualization capacity ofESCs. According to reports of immortalized cell lines, asimian virus 40 large T antigen (SV40T)-immortalizedESC line did not respond to E2 stimulation for PRexpression and prostaglandin production, possibly by

nonfunctional ERa [7], and another temperature-sensi-tive SV40T-immortalized ESC line showed an adequatedecidualization phenotype only with cAMP stimulation,even in the absence of progestin [11]. A recent hTERT-immortalized cell line showed a robust response to pro-gestin stimulation, but the synergistic effect of cAMP inthis cell line was not examined [9]. Another hTERT-immortalized line showed progestin and cAMP synergy,but in contrast to our clone, PR induction by E2 wasnot noted in this cell line [10]. The reasons for thesediscrepancies are not clear, but the differences inhuman ESC sources or E2-supplemented culture condi-tions for clone selection might affect the ability of eachcell line.Human ESCs respond to IL-1b through induction of

COX-2 expression and PGE2 production. In primaryhuman ESCs, COX-2 mRNA is induced by IL-1b in ap38 mitogen-activated protein kinase (p38MAPK)-dependent manner and this induction is diminished byprogesterone or 8-bromo-cAMP through p38MAPK

Figure 3 Inflammatory responses of KC02-44D cells to IL-1b. KC02-44D cells were cultured with the indicated combinations of 10-8 M E2,10-6 M MPA, and 0.125 mM dibutyryl cAMP in the absence or presence of IL-1b (1.0 ng/ml) for 7 days to observe their inflammatory responses.(a) Expression of COX-2 in KC02-44D cells at passage number 14 (RT-PCR analysis). b-Actin mRNA was used for normalization. (b) Expression ofPGE2 in KC02-44D cells at passage number 16 (ELISA). (c) Expression of IL-8 in KC02-44D cells at passage number 19 (ELISA). The data representthe mean ± SEM of triplicate measurements. ***P < 0.001 by Tukey’s test. Letter a indicate differences between the control and IL-1b for eachstimulate: p < 0.001.


of 9

inhibition [27], which is consistent with the resultsregarding the inflammatory responses of KC02-44Dcells. Further, we observed induction of IL-8 productionupon IL-1b stimulation. However, progestin and/orcAMP seemed to have no inhibitory effect on IL-8expression. A previous study using endometriotic tissuesshowed a similar observation, indicating the menstrualcycle phase-independent expression of IL-8 and proges-tin resistance of IL-1b-induced IL-8 production [28].However, the mechanism of progestin resistance of IL-8induction is currently unclear; a PR-dependent mechan-ism might be responsible. At least distinct responseswere observed for COX-2 and IL-8 induction by IL-1bin our cell line.In the menstrual cycle, estrogen induces PR expres-

sion in the proliferative phase, and a high level of PRexpression is observed in the late proliferative phase;this expression is downregulated according to the eleva-tion in the serum progesterone level, followed by theinitiation of decidualization in the secretory phase[29,30]. We have shown the regulation of PR expressionin this study. E2 strongly upregulated the expression ofthe PR, and the E2-induced PR expression was signifi-cantly downregulated after the stimulation with MPA(Figure 2c). Compared with paired ectopic endometrialspecimens, a drastic loss of PR-B expression has beenobserved in endometriotic tissues, resulting in progester-one resistance in endometriosis [31]. IL-1b-inducedinhibition of the decidualization capacity [24-26], activa-tion of cell proliferation, and downregulation of PR-Bexpression have been observed in primary ESCs [32].We confirmed the effects of IL-1b on these responses inKC02-44D cells.

ConclusionsWe established an immortalized human ESC line, KC02-44D, by introducing the hTERT gene into primary ESCs.This clone showed characteristics such as decidualiza-tion capacity and responses to IL-1b similar to primaryhuman ESCs. The KC02-44D cell line would be usefulto investigate the mechanisms of normal and pathologi-cal human reproductive processes.

Additional material

Additional file 1: Supplemental Figure S1. Induction of Prolactin andIGFBP-1 expression in KC02-44D cells.

Additional file 2: Supplemental Figure S2. Long-term maintenance ofKC02-44D cells

List of abbreviations usedBrdU: 5-bromo-2’-deoxyuridine; cAMP: 3’,5’-cyclic adenosine monophosphate;COX-2: cyclooxygenase-2; DCC-FBS: dextran-coated charcoal-treated fetalbovine serum; DMEM: Dulbecco’s modified Eagle’s medium; E2: estradiol;

Figure 4 IL-1b-induced downregulation of the decidualizationcapacity of KC02-44D cells. KC02-44D cells were pretreated withIL-1b (1.0 ng/ml) for 4 days and then stimulated with the indicatedcombinations of 10-8 M E2, 10-6 M MPA, and 0.125 mM dibutyrylcAMP for 7 days. (a) Expression of decidualization marker IGFBP-1 inKC02-44D cells at passage number 19 (ELISA). The data representthe mean ± SEM of triplicate measurements. *P < 0.05, *P < 0.01and ***P < 0.001 by Tukey’s test. (b) Expression of IGFBP-1 and thePR in KC02-44D cells at passage number 16 (Western blot analysis).b-Actin was used for normalization. † = nonspecific band.

Figure 5 Counteraction of decidualization stimuli-induced cellgrowth arrest by IL-1b. EdU assay was performed with KC02-44Dcells at passage number 15 pretreated with IL-1b (1.0 ng/ml) for 3days and then stimulated with the indicated combinations of 10-8

M E2, 10-6 M MPA, and 0.125 mM dibutyryl cAMP for 24 h. The EdUintensity was measured by an ArrayScan reader. The data representthe mean ± SEM of triplicate measurements. ***P < 0.001 by Tukey’stest.


of 9

http://www.biomedcentral.com/content/supplementary/1477-7827-9-104-S1.PPT

http://www.biomedcentral.com/content/supplementary/1477-7827-9-104-S2.PPT

EdU: 5-ethynyl-2’-deoxyuridine; ELISA: enzyme-linked immunosorbent assay;ESC: endometrial stromal cell; ERα: human estrogen receptor-alpha; hTERT:human telomerase reverse transcriptase; IGFBP-1: insulin-like growth factor-binding protein-1; IL: interleukin; LtV-hTERT: a lentivirus encoding hTERT;MPA: 6α-methyl-17α-hydroxy-progesterone acetate; PBS: phosphate-bufferedsaline; PFA: paraformaldehyde; PKA: protein kinase A; PGE2: prostaglandin E2;PR: progesterone receptor; RT-PCR: reverse transcription polymerase chainreaction; TBS-T: Tris-buffered saline containing 0.1% Tween-20; TNF-α: tumornecrosis factor-alpha; p38MAPK: p38 mitogen-activated protein kinase

AcknowledgementsWe thank Dr. H. Masuda for collecting the endometrial sample. We alsothank Dr K. Taniguchi for the helpful discussion and Ms. N. Aoyama for herexpert technical assistance. Editorial and publication support was providedby Editage.

Author details1Kamakura Research Laboratories, Chugai Pharmaceutical Co., Ltd., 200Kajiwara, Kamakura, Kanagawa 247-8530, Japan. 2Department of Obstetricsand Gynecology, Keio University School of Medicine, Tokyo 160-8582, Japan.

Authors’ contributionsThe work presented here was carried out in collaboration by all the authors.TM and TM defined the study concept. MY and TM designed the methodsand experiments, performed the laboratory experiments, analyzed the data,interpreted the results, and wrote the manuscript. TK codesigned theexperiments and coworked on the associated data collection and theirinterpretation. YA codesigned the experiments and participated in thediscussion, analyses, interpretation, and presentation of the study data. Allauthors read and approved the final manuscript.

Competing interestsMY, T. Mizuno, and YA are employees of Chugai Pharmaceutical Co. Ltd.,Japan. The authors have no other relevant affiliations or financialinvolvement with any organization or entity with a financial interest in orfinancial conflict with the subject matter or materials discussed in themanuscript apart from those disclosed.

Received: 18 March 2011 Accepted: 1 August 2011Published: 1 August 2011

References1. Ing NH, Spencer TE, Bazer FW: Estrogen enhances endometrial estrogen

receptor gene expression by a posttranscriptional mechanism in theovariectomized ewe. Biol Reprod 1996, 54:591-599.

2. Kurita T, Lee KJ, Cooke PS, Lydon JP, Cunha GR: Paracrine regulation ofepithelial progesterone receptor and lactoferrin by progesterone in themouse uterus. Biol Reprod 2000, 62:831-838.

3. Nephew KP, Long X, Osborne E, Burke KA, Ahluwalia A, Bigsby RM: Effect ofestradiol on estrogen receptor expression in rat uterine cell types. BiolReprod 2000, 62:168-177.

4. Gellersen B, Brosens J: Cyclic AMP and progesterone receptor cross-talkin human endometrium: a decidualizing affair. J Endocrinol 2003,178:357-372.

5. Bergqvist A, Bruse C, Carlberg M, Carlström K: Interleukin 1β, interleukin-6,and tumor necrosis factor-α in endometriotic tissue and inendometrium. Fertil Steril 2001, 75:489-495.

6. Hudelist G, Lass H, Keckstein J, Walter I, Wieser F, Wenzl R, Mueller R,Czerwenka K, Kubista E, Singer CF: Interleukin 1α and tissue-lytic matrixmetalloproteinase-1 are elevated in ectopic endometrium of patientswith endometriosis. Hum Reprod 2005, 20:1695-1701.

7. Chapdelaine P, Kang J, Boucher-Kovalik S, Caron N, Tremblay JP, Fortier MA:Decidualization and maintenance of a functional prostaglandin systemin human endometrial cell lines following transformation with SV40large T antigen. Mol Hum Reprod 2006, 12:309-319.

8. Barbier CS, Becker KA, Troester MA, Kaufman DG: Expression of exogenoushuman telomerase in cultures of endometrial stromal cells does notalter their hormone responsiveness. Biol Reprod 2005, 73:106-114.

9. Krikun G, Mor G, Alvero A, Guller S, Schatz F, Sapi E, Rahman M, Caze R,Qumsiyeh M, Lockwood CJ: A novel immortalized human endometrial

stromal cell line with normal progestational response. Endocrinology2004, 145:2291-2296.

10. Samalecos A, Reimann K, Wittmann S, Schulte HM, Brosens JJ,Bamberger AM, Gellersen B: Characterization of a novel telomerase-immortalized human endometrial stromal cell line, St-T1b. Reprod BiolEndocrinol 2009, 7:76.

11. Tamura K, Yoshie M, Hara T, Isaka K, Kogo H: Involvement of stathmin inproliferation and differentiation of immortalized human endometrialstromal cells. J Reprod Dev 2007, 53:525-533.

12. Nagashima T, Maruyama T, Uchida H, Kajitani T, Arase T, Ono M, Oda H,Kagami M, Masuda H, Nishikawa S, Asada H, Yoshimura Y: Activation ofSRC kinase and phosphorylation of signal transducer and activator oftranscription-5 are required for decidual transformation of humanendometrial stromal cells. Endocrinology 2008, 149:1227-1234.

13. Brosens JJ, Hayashi N, White JO: Progesterone receptor regulates decidualprolactin expression in differentiating human endometrial stromal cells.Endocrinology 1999, 140:4809-4820.

14. Borthwick JM, Charnock-Jones DS, Tom BD, Hull ML, Teirney R, Phillips SC,Smith SK: Determination of the transcript profile of humanendometrium. Mol Hum Reprod 2003, 9:19-33.

15. Brandenberger AW, Lebovic DI, Tee MK, Ryan IP, Tseng JF, Jaffe RB,Taylor RN: Oestrogen receptor (ER)-α and ER-β isoforms in normalendometrial and endometriosis-derived stromal cells. Mol Hum Reprod1999, 5:651-655.

16. Classen-Linke I, Kusche M, Knauthe R, Beier HM: Establishment of a humanendometrial cell culture system and characterization of its polarizedhormone responsive epithelial cells. Cell Tissue Res 1997, 287:171-185.

17. Milgrom E, Thi L, Atger M, Baulieu EE: Mechanisms regulating theconcentration and the conformation of progesterone receptor(s) in theuterus. J Biol Chem 1973, 248:6366-6374.

18. Saegusa M, Okayasu I: Changes in expression of estrogen receptors α andβ in relation to progesterone receptor and pS2 status in normal andmalignant endometrium. Jpn J Cancer Res 2000, 91:510-518.

19. Shiozawa T, Li SF, Nakayama K, Nikaido T, Fujii S: Relationship between theexpression of cyclins/cyclin-dependent kinases and sex-steroidreceptors/Ki67 in normal human endometrial glands and stroma duringthe menstrual cycle. Mol Hum Reprod 1996, 2:745-752.

20. Cheong YC, Shelton JB, Laird SM, Richmond M, Kudesia G, Li TC,Ledger WL: IL-1, IL-6 and TNF-α concentrations in the peritoneal fluid ofwomen with pelvic adhesions. Hum Reprod 2002, 17:69-75.

21. Keenan JA, Chen TT, Chadwell NL, Torry DS, Caudle MR: IL-1β, TNF-α, andIL-2 in peritoneal fluid and macrophage-conditioned media of womenwith endometriosis. Am J Reprod Immunol 1995, 34:381-385.

22. Mori H, Sawairi M, Nakagawa M, Itoh N, Wada K, Tamaya T: Peritoneal fluidinterleukin-1β and tumor necrosis factor in patients with benigngynecologic disease. Am J Reprod Immunol 1991, 26:62-67.

23. Klemmt PA, Carver JG, Kennedy SH, Koninckx PR, Mardon HJ: Stromal cellsfrom endometriotic lesions and endometrium from women withendometriosis have reduced decidualization capacity. Fertil Steril 2006,85:564-572.

24. Frank GR, Brar AK, Jikihara H, Cedars MI, Handwerger S: Interleukin-1β andthe endometrium: an inhibitor of stromal cell differentiation andpossible autoregulator of decidualization in humans. Biol Reprod 1995,52:184-191.

25. Kariya M, Kanzaki H, Takakura K, Imai K, Okamoto N, Emi N, Kariya Y, Mori T:Interleukin-1 inhibits in vitro decidualization of human endometrialstromal cells. J Clin Endocrinol Metab 1991, 73:1170-1174.

26. Mizuno K, Tanaka T, Umesaki N, Ogita S: Inhibition of cAMP-mediateddecidualization in human endometrial stromal cells by IL-1β andlaminin. Horm Metab Res 1999, 31:307-310.

27. Yoshino O, Osuga Y, Hirota Y, Koga K, Hirata T, Yano T, Ayabe T,Tsutsumi O, Taketani Y: Endometrial stromal cells undergoingdecidualization down-regulate their properties to produceproinflammatory cytokines in response to interleukin-1β via reducedp38 mitogen-activated protein kinase phosphorylation. J Clin EndocrinolMetab 2003, 88:2236-2241.

28. Akoum A, Lawson C, McColl S, Villeneuve M: Ectopic endometrial cellsexpress high concentrations of interleukin (IL)-8 in vivo regardless of themenstrual cycle phase and respond to oestradiol by up-regulating IL-1-induced IL-8 expression in vitro. Mol Hum Reprod 2001, 7:859-866.


of 9

http://www.ncbi.nlm.nih.gov/pubmed/8835380?dopt=Abstract















































































29. Mote PA, Balleine RL, McGowan EM, Clarke CL: Heterogeneity ofprogesterone receptors A and B expression in human endometrialglands and stroma. Hum Reprod 2000, 15:48-56.

30. Stricker R, Eberhart R, Chevailler MC, Quinn FA, Bischof P: Establishment ofdetailed reference values for luteinizing hormone, follicle stimulatinghormone, estradiol, and progesterone during different phases of themenstrual cycle on the Abbott ARCHITECT analyzer. Clin Chem Lab Med2006, 44:883-887.

31. Attia GR, Zeitoun K, Edwards D, Johns A, Carr BR, Bulun SE: Progesteronereceptor isoform A but not B is expressed in endometriosis. J ClinEndocrinol Metab 2000, 85:2897-2902.

32. Igarashi T, Nakamura Y, Ochiai N, Muto S, Kawamura H, Nakagawa K,Uesato T, Ryo Y: Dioxin may regulate progesterone action and promotedevelopment of endometriosis. J Jap Soc Endometriosis 2007, 28:26-32, (inJapanese).

doi:10.1186/1477-7827-9-104Cite this article as: Yuhki et al.: Establishment of an immortalizedhuman endometrial stromal cell line with functional responses toovarian stimuli. Reproductive Biology and Endocrinology 2011 9:104.

Submit your next manuscript to BioMed Centraland take full advantage of:

• Convenient online submission

• Thorough peer review

• No space constraints or color figure charges

• Immediate publication on acceptance

• Inclusion in PubMed, CAS, Scopus and Google Scholar

• Research which is freely available for redistribution

Submit your manuscript at www.biomedcentral.com/submit


of 9










METHODOLOGY Open Access Establishment of an immortalized ... · Establishment of an immortalized...

Documents

Transcript of METHODOLOGY Open Access Establishment of an immortalized ... · Establishment of an immortalized...